CD47 Is Necessary for Inhibition of Nitric Oxide-stimulated Vascular Cell Responses by Thrombospondin-1

Jeff S Isenberg,Lisa A Ridnour,Julie Dimitry,William A Frazier,David A Wink,David D Roberts

doi:10.1074/jbc.m605040200

Abstract

CD36 is necessary for inhibition of some angiogenic responses by the matricellular glycoprotein thrombospondin-1 and is therefore assumed to be the receptor that mediates its anti-angiogenic activities. Although ligation of CD36 by antibodies, recombinant type 1 repeats of thrombospondin-1, or CD36-binding peptides was sufficient to inhibit nitric oxide (NO)-stimulated responses in both endothelial and vascular smooth muscle cells, picomolar concentrations of native thrombospondin-1 similarly inhibited NO signaling in vascular cells from wild-type and CD36-null mice. Ligation of the thrombospondin-1 receptor CD47 by recombinant C-terminal regions of thrombospondin-1, thrombospondin-1 peptides, or CD47 antibodies was also sufficient to inhibit NO-stimulated phenotypic responses and cGMP signaling in vascular cells. Thrombospondin-1 did not inhibit NO signaling in CD47-null vascular cells or NO-stimulated vascular outgrowth from CD47-null muscle explants in three-dimensional cultures. Furthermore, the CD36-binding domain of thrombospondin-1 and anti-angiogenic peptides derived from this domain failed to inhibit NO signaling in CD47-null cells. Therefore, ligation of either CD36 or CD47 is sufficient to inhibit NO-stimulated vascular cell responses and cGMP signaling, but only CD47 is necessary for this activity of thrombospondin-1 at physiological concentrations.

Highlights

Thrombospondin-1 (TSP1)2 is a secreted glycoprotein that plays a relatively minor role in development of the murine vascular system [1, 2], but its regulated appearance in the extracellular matrix plays important roles in responses to acute injury and in several chronic disease states in the adult
Some evidence indicates that TSP1 inhibits angiogenesis through ␤1 integrins, CD47, LRP/calreticulin, and heparan sulfate proteoglycans (14, 19 –21), but the necessity of these receptors for TSP1 to inhibit angiogenesis has not been confirmed in the respective receptor-null mice
Peptide Ligands of Three TSP1 Receptors Inhibit Explant Angiogenic Responses—As previously reported [22], sustained exposure to exogenous nitric oxide (NO) released by the donor DETA/NO stimulated vascular outgrowth from muscle explants in three-dimensional collagen cultures to a greater extent in those from TSP1-null when compared with WT mice (Fig. 1A, controls)

Summary

EXPERIMENTAL PROCEDURES

Chromatography (Pierce) from conditioned media of the respective hybridoma (American Type Culture Collection). Handling and care of animals was in compliance with the guidelines established by the Animal Care and

Cancer Institute and of Washington

RESULTS

Necessary for Inhibition of NO

DISCUSSION